Postnatal Growth of the Human Pons: a Morphometric and Immunohistochemical Analysis

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Title Postnatal growth of the human pons: a morphometric and immunohistochemical analysis.

Permalink https://escholarship.org/uc/item/9579m17k

Journal The Journal of comparative neurology, 523(3)

ISSN 0021-9967

Authors Tate, Matthew C Lindquist, Robert A Nguyen, Thuhien et al.

Publication Date 2015-02-01

DOI 10.1002/cne.23690

Peer reviewed

eScholarship.org Powered by the California Digital Library University of California RESEARCH ARTICLE

Postnatal Growth of the Human Pons: A Morphometric and Immunohistochemical Analysis

Matthew C. Tate,1,2† Robert A. Lindquist,1,3,4† Thuhien Nguyen,1,2 Nader Sanai,5 A. James Barkovich,6 Eric J. Huang,3,7 David H. Rowitch,1,2,3,8 and Arturo Alvarez-Buylla1,2,3* 1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California – San Francisco, San Francisco, CA 94143 2Department of Neurological Surgery, University of California – San Francisco, San Francisco, CA 94143 3Neuroscience Graduate Program, University of California – San Francisco, San Francisco, CA 94143 4Medical Scientist Training Program, School of Medicine, University of California – San Francisco, San Francisco, CA 94143 5Department of Neurological Surgery, Barrow Neurological Institute, Phoenix, AZ 85013 6Department of Radiology and Biomedical Imaging, Section of Pediatric Neuroradiology, University of California – San Francisco, San Francisco, CA 94143 7Department of Pathology, University of California – San Francisco, San Francisco, CA 94143 8Howard Hughes Medical Institute and Department of Pediatrics, University of California – San Francisco, San Francisco, CA 94143

ABSTRACT majority of proliferative cells in the postnatal pons Despite its critical importance to global brain function, the expressed the transcription factor Olig2, suggesting an oli- postnatal development of the human pons remains poorly godendrocyte lineage. The proportion of proliferating cells understood. In the present study, we first performed mag- that were Olig21 was similar through the first 7 months netic resonance imaging (MRI)-based morphometric analy- of life and between basis and tegmentum. The number of ses of the postnatal human pons (0–18 years; n 5 6– Ki671 cells declined dramatically from birth to 7 months 14/timepoint). Pons volume increased 6-fold from birth to and further decreased by 3 years, with a small number of 5 years, followed by continued slower growth throughout Ki671 cells observed throughout childhood. In addition, childhood. The observed growth was primarily due to two populations of vimentin/nestin-expressing cells were expansion of the basis pontis. T2-based MRI analysis sug- identified: a dorsal group near the ventricular surface, gests that this growth is linked to increased myelination, which persists throughout childhood, and a parenchymal and histological analysis of myelin basic protein in human population that diminishes by 7 months and was not evi- postmortem specimens confirmed a dramatic increase in dent later in childhood. Together, our data reveal remark- myelination during infancy. Analysis of cellular proliferation able postnatal growth in the ventral pons, particularly revealed many Ki671 cells during the first 7 months of during infancy when cells are most proliferative and myeli- life, particularly during the first month, where proliferation nation increases. J. Comp. Neurol. 523:449–462, 2015. was increased in the basis relative to tegmentum. The VC 2014 Wiley Periodicals, Inc.

INDEXING TERMS: brainstem; pediatric; development; basis; pontine glioma; nif-0000-00217; SciRes_000114; AB_304558; AB_2109815; AB_442102; AB_396287; AB_92396; AB_91107; AB_291466; AB_2336877; AB_2336878; AB_261856; AB_2304493

The ﬁrst two authors contributed equally to this work. Center Way RMB-1038, Box 0525, San Francisco, CA 94143-0525. E-mail: [email protected] Grant sponsor: National Institutes of Health; Grant number: NS28478; Grant sponsor: John G. Bowes Research Fund (to A.A.-B.); Grant num- Received June 23, 2014; Revised September 30, 2014; ber: NRSA 1F32NS067889-01A1 (to M.T.); Grant sponsor: Pediatric Accepted October 6, 2014. Brain Tumor Foundation (to A.A.-B., D.H.R.), University of California; DOI 10.1002/cne.23690 Grant number: MRPI #142675 (to E.J.H.); Grant sponsor: D.H.R. is an Published online October 10, 2014 in Wiley Online Library HHMI Investigator. (wileyonlinelibrary.com) *CORRESPONDENCE TO: Arturo Alvarez-Buylla, PhD, University of California, San Francisco, Department of Neurological Surgery, 35 Medical

VC 2014 Wiley Periodicals, Inc.

The Journal of Comparative Neurology | Research in Systems Neuroscience 523:449–462 (2015) 449 M.C. Tate et al.

During embryogenesis, the pons and cerebellum are formed from the metencephalon, which is derived from the rostral hindbrain (rhombencephalon) (Barkovich et al., 2009). In humans, pontine nuclei are derived from a stream of cells migrating from the rhombic lip termed the corpus pontobulbare between 8 and 20 weeks gestation (Essick, 1912). While a number of studies describe the development of the fetal pons (Nozaki et al., 1992; Fischbein et al., 1996; Hatta et al., 2007), less is known about postnatal pontine growth in humans, particularly at the cellular level. An improved understanding of the dynamics of growth and development of the human pons, particularly postnatal stem/ progenitor populations, may provide insight into the development of this critical brain region and into abnormalities of the pons such as developmental malforma- tions and pediatric gliomas (Barkovich et al., 2009). In this study, we quantified spatial and temporal growth profiles of the human pons throughout childhood using magnetic resonance imaging (MRI)-based morphometry. In addition, we examined cell proliferation and expression of progenitor markers as a function of age and location within the pons.

MATERIALS AND METHODS Figure 1. Brainstem morphometric measurements. The upper panel Brainstem morphometry (A) is an example mid-sagittal T1-weighted MRI showing the basis pontis (yellow), pontine tegmentum (red), and medulla (blue). All brain MRI studies were approved by the University Dashed lines illustrate the axial sections extending from proximal of California, San Francisco (UCSF) Committee on pons to distal medulla that were used to estimate volumes. For Human Research. MRI scans obtained between 2005 each cross-section, the relevant brainstem subregions (tegmentum, and 2010, for which there were no intracranial abnor- basis, and medulla) were traced to determine the volume. The malities (as noted by the official report of the neurora- lower panels illustrate the tracing of axial images through mid-pons (B, left) and mid-medulla (C, right) levels. D 5 dorsal, V 5 ventral, diologist who interpreted the scan as part of routine R 5 rostral, C 5 caudal. clinical care), were included in the study (n 5 123). For volumetric assessments (total pons, medulla, pontine tegmentum, basis pontis), T1-weighted sequences were The human pons, part of the brainstem, is an impor- evaluated. In addition, average T2 values for both basis tant relay for sensory and motor information from the and tegmentum were measured from mid-pons axial forebrain to the cerebellum. In addition, the pons plays images, and the ratios of T2 intensities were used to essential roles in respiration, sleep, swallowing, eye estimate myelination in these two regions (modification movement, hearing, facial movement/sensation, pos- of a method described by Abe et al., 2004). For each ture, and maintenance of consciousness (Saladin, MRI, consecutive axial images extending from rostral 2012). Anatomically, the pons can be divided into three pons (at midbrain-pons junction, just caudal to cerebral regions: tectum, tegmentum, and basis. The tectum is peduncles) to distal medulla (cervicomedullary junction) the most dorsal component and forms the roof of the were analyzed. The areas (in mm2) of regions of interest 4th ventricle. The tegmentum is also located dorsally (pontine tegmentum, pontine basis, medulla) were man- within the pons and contains important autonomic ually traced for each axial image and the volume (in structures, as well as nuclei of cranial nerves V–VIII, mm3) estimated using measurement tools from Philips which control various facial movements and sensation. iSite Enterprise (v. 3.6) radiology software (Fig. 1). Age The ventral portion of the pons is occupied by the basis groups evaluated were age 0 (within 3 days of birth, pontis. Pontine nuclei within the basis receive inputs normal gestational age), 3 months, 6 months, 9 from descending corticopontine fibers and project to months, 1 year, 3 years, 5 years, 7 years, 9 years, 11 the cerebellum via the middle cerebellar peduncle. years, 13 years, and 18 years (n 5 123). Average

450 The Journal of Comparative Neurology | Research in Systems Neuroscience Human postnatal pons development

TABLE 1. List of Human Specimens

Specimen Postnatal Gestational APGAR Postmortem Year of Number Age Length (wks) (1,5 min) Gender Diagnosis Interval (hrs) Autopsy Source 1 1 day 38 1/7 1,0 F Pulmonary failure 72 2011 1 2 1 mo 35 6/7 8,8 M Cardiac anomaly 36 2008 1 3 2 mo 37 5,6 M Hepatic failure 24 2011 1 4 7 mo 31 4/7 — M Cardiac anomaly 24 2011 1 5 1.5 yr — — F Sepsis 29 2007 1 6 3 yr — — M Asphyxia 16 2009 2 7 7 yr — — F Lymphoma 2 2007 1 8 8 yr — — M Trauma 16 2009 2 9 13 yr — — M Trauma 15 2009 2 10 13.5 yr — — M Trauma 19 2010 2

1 UCSF Dept. Pathology. 2 NICHD Brain and Tissue Bank. volumetric growth rates for basis, tegmentum, and Immunohistochemistry medulla (mm3/mo) were calculated between age After rinses in TNT wash buffer (13 phosphate- groups (0–3 months, 3–6 months, 6 months to 1 year, buffered saline, 0.05% Triton X-100), microwave or water 1–3 years, 3–5 years, 5–7 years, 7–18 years) by calcu- bath antigen retrieval was performed for all sections in lating the difference in volumetric averages of the two 0.01M citrate buffer (pH 6.0) at 95Cfor10minutes,fol- age groups (in mm3) and dividing by the time between lowed by a 20-minute cooling period. After rinsing in TNT, ages (in months). slides were incubated with 1–2% H2O2 for 30–60 minutes at room temperature to block endogenous peroxidase Human brainstem specimens and tissue activity. Slides were then incubated in TNB blocking solu- preparation tion (0.1M Tris-HCl, pH 7.5, 0.15M NaCl, 0.5% blocking Samples of postmortem human pons were collected reagent from PerkinElmer, Waltham, MA) for 30 minutes from two sources (n 5 10, Table 1). Specimens at room temperature, followed by overnight incubation in obtained at autopsy within the UCSF Department of primary antibodies (see Table 2 for details of primary antibodies used). Sections were then incubated for 90 Pathology as part of a standard operating procedure minutes in biotinylated secondary antibodies (dilution were transferred to 4% paraformaldehyde within 72 1:500; Jackson ImmunoResearch, West Grove, PA) for hours of death. All tissues were collected in accordance Ki67, nestin, mouse Olig2, and anti-myelin basic protein with the UCSF Committee on Human Research. Addi- (MBP) primary antibodies and/or direct fluorophore- tional specimens harvested <24 hours postmortem and conjugated secondary antibodies (dilution 1:500; Life stored in 10% formalin fixative were obtained from the Technologies, Grand Island, NY) for vimentin, rabbit Olig2 National Institute of Child Health and Human Develop- antibodies, and GFAP primary antibodies. All secondary ment Brain and Tissue Bank for Developmental Disor- antibodies were diluted in TNB along with DAPI at ders at the University of Maryland, Baltimore (contract 1:10,000 dilution. Biotinylated sections were then incu- HHSN275200900011C, ref. N01-HD-9-0011, RRID: nif- bated in streptavidin-horseradish peroxidase (PerkinElmer) 0000-00217). Fixative storage intervals for specimens for 30 minutes, followed by application of DAB Peroxidase included in the analysis ranged from 0–3 years. For Substrate Kit (MBP, Vector Labs, Burlingame, CA) for 7.5 quality control purposes, autopsy specimens that were minutes or TSA fluorescent amplification (nestin, Ki67, grossly damaged or did not stain for the nuclear marker mouse vimentin) for 4–5 minutes, which uses fluorescent DAPI were excluded. All samples included in the analy- conversion of tyramide substrates (PerkinElmer). For sis were derived from patients with no evidence of double-labeling studies, a similar protocol was employed, intracranial abnormalities. with the exception of administering both a biotinylated Axial blocks of 5 mm thickness were cryoprotected and direct fluorophore-conjugated secondary antibody in 30% sucrose solution, snap-frozen in OCT compound simultaneously in the secondary antibody incubation step. (Tissue-Tek, Torrance, CA) using dry ice, and placed in a –80C freezer for equilibration. Axial 18–20-lm thick sections were collected using a standard cryotome and Antibody characterization mounted on glass slides (Superfrost Plus, Fischer Scien- The details of the primary antibodies used in the tific, Waltham, MA). study are included in Table 2. The chicken polyclonal

The Journal of Comparative Neurology | Research in Systems Neuroscience 451 M.C. Tate et al.

TABLE 2. Primary Antibodies Used

Source, Species/Clonality, Tyramide Antigen Immunogen Catalog no., RRID Dilution Amplification GFAP bovine full length protein Abcam, chicken polyclonal, Cat. no. ab4674, 1:500 N RRID:AB_304558 GFAP purified GFAP from porcine spinal EMD Millipore, mouse monoclonal IgG1 (clone GA5), 1:1,000 N cord Cat. no. MAB360, RRID:AB_2109815 Ki67 Ki67 motif-containing cDNA Leica Microsystems, rabbit polyclonal, Cat. no. NCL- 1:1,000 Y fragment Ki67p, RRID:AB_442102 Ki67 human Ki67 BD Biosciences, mouse monoclonal IgG1j (clone 1:200 Y B56), Cat. no. 556003, RRID:AB_396287 MBP human myelin basic protein EMD Millipore, rabbit polyclonal, Cat. no. AB980, 1:500 N* RRID:AB_92396 Nestin human nestin cDNA fragment EMD Millipore, rabbit polyclonal, Cat. no. AB5922, 1:500 Y RRID:AB_91107 Nestin human nestin cDNA fragment Covance Research Products, mouse monoclonal IgG1 1:500 Y (clone 2C1 3B9), Cat. no. MMS-570P-100, RRID:AB_291466 Olig2 fusion protein containing N-terminus CD Stiles/Harvard, rabbit polyclonal, Cat. no. DF308, 1:1,000 N of mouse Olig2 RRID: AB_2336877 Olig2 fusion protein containing N-terminus CD Stiles/Harvard, mouse monoclonal IgG, Cat. no. 1:500 Y of mouse Olig2 TV73–1C10, RRID: AB_2336878 Vimentin human thymic nuclear extract Sigma-Aldrich, mouse monoclonal IgM (clone LN-6), 1:200 N Cat. no. V2258, RRID:AB_261856 Vimentin bovine eye lens protein Dako, mouse monoclonal IgG2a (clone Vim 3B4), Cat. 1:100 Y no. M7020, RRID:AB_2304493

*Chromogenic DAB amplification. antibody against GFAP was characterized by the manu- blot. The rabbit anti-Olig2 antibody was characterized facturer in a western blot analysis of brain tissue lysate, via western blot of human normal human cortex and yielding bands at 55 kDa and 48 kDa, as well as by human glioma lysates (38 kDa band), as well as colocal- flow cytometry of human brain cells. The mouse mono- ization with PDGFRa-positive cells in human fetal white clonal GFAP antibody was previously characterized by matter using double-label immunohistochemistry (Ligon western blot of human glioma cell lines, yielding a band et al., 2004). Specificity of the mouse monoclonal anti- at 51 kDa, while no band was detected in human RD Olig2 antibody was demonstrated by a lack of staining cells that lack GFAP (Debus et al., 1983). The rabbit in Olig2-null brain tissue immunohistochemistry (Ligon polyclonal Ki67 antibody was characterized by the man- et al., 2006). The mouse IgM anti-vimentin antibody ufacturer to localize to the nucleus of normal and neo- labeled the expected 60 kDa band in western blot of plastic human tissue. The mouse monoclonal Ki67 human sarcoma cells (Stathopoulos et al., 1989), and antibody was tested in western blot of proliferating western blot of human HeLa cell lysate using the cells by the manufacturer, yielding bands at 395 and mouse IgG anti-valentine antibody recognized a band at 345 kDa, and immunohistochemical staining in human 50 kDa (Bohn et al., 1992). tonsil yielded similar results to MIB-1 antibody that also For all antibodies listed, the staining protocol was reacts with Ki67. For the rabbit polyclonal against optimized in single-labeling studies after varying antigen human myelin basic protein, manufacturer data indi- retrieval (6 citrate antigen retrieval), primary antibody cates western blot detected bands in mouse brain concentration (dilution series), and secondary antibody lysate (14, 17, 18, and 21 kDa), and a recent study method (fluorophore-conjugated secondary antibody demonstrated colocalization of this antibody with other versus biotinylated with TSA amplification). Given the myelin components using human adult brain immuno- paucity of data in the human pons for expression of the histochemistry (Roig et al., 2010). Western blot analysis antigens of interest (Ki67, Olig2, vimentin, nestin), two of the rabbit polyclonal anti-nestin antibody in two dis- independent primary antibodies, typically different host tinct human glioma cell line lysates revealed a single species and clonality (see Table 2), were used for each band between 220 and 240 kDa (Messam et al., 2000). antigen to ensure specificity/sensitivity of the antibod- Likewise, manufacturer information for the mouse ies. Only those antibodies that produced a spatial and monoclonal anti-nestin antibody indicates human speci- temporal distribution that could be confirmed with at ficity with a 220–240 kDa band detected by western least one other antibody were used for the study. For

452 The Journal of Comparative Neurology | Research in Systems Neuroscience Human postnatal pons development

myelin immunohistochemistry with anti-MBP, a similar For each section, the number of Ki671 and Ki671/ spatial and temporal pattern of myelin staining was Olig21 cells were counted by a blinded observer, and confirmed with Luxol Fast Blue. In addition, for both sin- the percentage of double-positive cells (Ki671Olig21/ gle- and double-label experiments, parallel sections Ki671) was calculated for tegmentum and basis at 0–1 incubated without primary antibodies were performed months and 2–7 months. as negative controls. Using this method, the observed nestin and vimentin pattern lining the fourth ventricle Brightfield imaging of myelin stains within the dorsal pons was in agreement with a recent Tissue sections were labeled with anti-MBP antibody study on the human pons (Monje et al., 2011). While and detected by DAB staining as described above, then the lack of previous reports on Olig2 and Ki67 distribu- counterstained with Gill’s hematoxylin #3 (Polysciences, tion in the developing human pons prevent direct com- Warrington, PA). parison, a consistent spatial and temporal distribution Brightfield images were acquired on a DMI4000B for each antigen was confirmed with multiple antibod- microscope with DFC295 color camera (Leica), using ies. In addition, the Olig2 and Ki67 antibodies used in LAS AF software to perform an automated tile scan of this study have been previously used to detect these entire hemisections under a 53 objective, and to cap- antigens in human brain material (Ligon et al., 2004; ture higher-resolution images of specific regions under Ligon et al., 2006; Quinones-Hinojosa et al., 2006). a203 objective.

Assessment of cell proliferation Analysis For quantitation and mapping of cell proliferation, A one-way analysis of variance (ANOVA) was used to mid-pons axial hemisections immunostained for Ki67 evaluate T2 ratio data and total proliferation levels as a were tiled at 103 magnification on an epifluorescence function of time. For regional analysis of proliferation microscope using a Stereo Investigator automated and percentage of proliferating cells that were Olig21, image capture system (MBF Bioscience, Williston, VT; a two-way ANOVA was performed to evaluate the RRID: SciRes_000114). Offline, all proliferating cells importance of age, location, and age-location interac- were marked for subsequent analysis by a blinded tion on the dependent variable. Tukey post-hoc compar- investigator. Criteria for inclusion as a positive Ki67 cell isons were used to examine within- and between-group included clear nuclear staining of Ki67, colocalization differences for each ANOVA. P < 0.05 was considered with the nuclear marker DAPI, and lack of nonspecific significant for all statistical tests. staining in the same distribution. After defining tegmentum and basis contours for each hemisection, average cell density (# Ki671 cells/mm2) for total pons, teg- RESULTS mentum, and basis was tabulated. Specimens were The human pons expands its volume 6-fold grouped into one of five age groups: I (1 day, 1 month), during postnatal child development II (2 months, 7 months), III (1.5 years, 3 years), IV (7 We first compared the postnatal growth of the years, 8 years), and V (13 years, 13.5 years). In addi- medulla and pons using MRI scans from children rang- tion, spatial distribution maps for tegmentum and basis ing in age from birth to 18 years of age (n 5 10–15/ as a function of postnatal age were plotted using Ster- timepoint). We subdivided the pons into dorsal (tegmen- eoInvestigator software. tum) and ventral (basis) regions. Figure 1 illustrates the areas studied in the sagittal and axial planes. Volumet- Olig2/Ki67 quantification ric analysis of the postnatal human pons revealed a Confocal images were captured on a Leica TCS SP5 dramatic growth over the first several years of life, with AOBS microscope (Leica Microsystems, Buffalo Grove, a tripling of the total pons volume in the first 6 months IL) using a 203 objective with 2.723 optical zoom to a (average growth rate of 532.9 mm3/mo), and an addi- resolution of 0.279 lm per pixel. Images were collected tional doubling between the ages of 6 months and 5 by automated acquisition of 0.08 mm2 fields containing years (rate 5 106.1 mm3/mo) (Fig. 2A). Between 5 only basis or only tegmentum. Between 30 and 200 and 18 years there was continued growth of the pons, fields were collected per region depending on the spec- though at a more modest rate (13.7 mm3/mo). In con- imen size. Images were collected at four wavelengths trast to the pons, the volume of the medulla showed corresponding to DAPI (405 nm laser), Olig2 (Alexa less expansion over the same time periods, with growth 488/488 nm laser), and Ki67 (Cy3/543 nm laser). In rates of 118.7, 19.6, and –0.2 mm3/mo over the 0–6 addition, an unstained channel (633 laser) was col- months, 6 months to 5 years, and 5–18 years time lected to identify regions of nonspecific fluorescence. periods, respectively.

The Journal of Comparative Neurology | Research in Systems Neuroscience 453 M.C. Tate et al.

Figure 2. Brainstem volume measurements. A: Volume of pons and medulla (mm3) as a function of postnatal age demonstrates robust and sustained growth in the pons compared to the medulla. B: Comparison of basis versus tegmentum volumes over time demonstrates that the majority of growth observed in the pons occurs in the basis. C: Analysis of the ratio of pons basis to tegmentum volume, illustrating increasing ratio over the first 6 months followed by stabilization for the remainder of childhood. D: In the basis pontis, volumetric growth rates are highest in the first 3 months, followed by decreased but steady growth up to 1 year that subsequently declines to near zero growth by 18 years. In contrast, the medulla and tegmentum growth rates rapidly declined by 3–6 months and 6 months to 1 year, respectively.

Regional analysis showed that the growth of the pons lination (Abe et al., 2004). The ratio of T2 intensity wasprimarilyduetoadramaticincreaseinthevolumeof between basis and tegmentum (Fig. 3A) decreased sig- the basis as compared to the tegmentum (Fig. 2B). Consist- nificantly throughout childhood (1-way ANOVA; F10,90 5 ent with this preferential growth of the basis, the ratio of 26.273, P < 0.001), ranging from 1.14 at birth to 0.85 basis to tegmentum volume increased from 1.5 at birth to at age 18. Tukey post-hoc analysis revealed that the T2 2.0 at 3–6 months and stabilized at 2.3–2.7 for the remain- ratio at birth was significantly higher than all other time- der of childhood (Fig. 2C). The average volumetric growth points (P < 0.001) and the ratio at 3 months was signifi- rates of the pontine basis as a function of postnatal age cantly higher than 6 months, 9 months, and 1 year (P < (Fig. 2D) revealed three phases of growth. In the first 3 0.01). The T2 ratio was not statistically different between months of life, the basis dramatically expanded in volume, any other timepoints between 6 months and 18 years. with a growth rate of 500 mm3/mo. Between 3 months These observations suggest that the dramatic growth of and 1 year, the expansion rate decreased to 225 mm3/mo the basis during the first phase may be due in part to and remained constant over this interval. After 1 year, the increased myelination. Because our T2 MRI data sug- growth rate gradually declined to near zero from 7 years gested a relative increase in myelination of the basis dur- onward. In contrast, medulla and tegmentum growth rates ing the first 6 months, we performed DAB-based had dramatically declined by 3–6 months and 6 months to immunostaining of myelin basic protein in pons sections 1 year, respectively. (Fig. 3B–J). We observed an increase in the extent of We next compared the T2 intensity signal in our MRI myelination from 1 day to 7 months, clearly visible as a section analysis. T2 is a reflection of water content in progressive thickening of myelinated tracts in the basis, the tissue and is inversely related to the amount of mye- including the longitudinal fibers of the corticospinal tract

454 The Journal of Comparative Neurology | Research in Systems Neuroscience Human postnatal pons development

Figure 3. Myelination in the postnatal basis pontis. A: Quantification of the ratio of T2 signal in the basis relative to tegmentum, which is inversely correlated with the degree of myelination in the basis, indicating that myelination coincides with the observed early postnatal volumetric growth of the basis (Fig. 2). One-way ANOVA demonstrated a significant effect of age on T2 ratio (F10,90 5 P < 0.001) and post- hoc Tukey comparison revealed a significant difference between age 0 and all other ages (*P < 0.05) and between 3 months and 6 months, 9 months, and 1 year (#P < 0.05). B: Immunolabeling of myelin basic protein in pons hemisections (counterstained with hematoxylin) reveals progress of myelination during early childhood (1 day to 3 years). Samples aged older than 3 years were similar to 3-year samples (data not shown). B0: Schematic shows location of white matter tracts running through pontine nuclei (PN) and corticospinal tract (CST), shown at higher magnification in C–J. C–J: Fibers in both regions undergo significant increase of myelination from birth to 7 months and more modest increase from 7 months to 3 years.

(CST) and the horizontally oriented fibers within the pon- Spatial and temporal profile of proliferation tine nuclei (PN). Myelination increased further from 7 in the postnatal human pons months to 3 years of age. Myelin distribution at ages The dramatic growth of the pons during postnatal older than 3 years was similar to 3-year samples, and life, and in particular of the basis, could be due to the similar results were obtained by labeling myelin with addition of new cells, growth and addition of cellular Luxol Fast Blue dye (data not shown). processes, or both. The T2 MRI analysis and MBP

The Journal of Comparative Neurology | Research in Systems Neuroscience 455 M.C. Tate et al.

Figure 4. Temporal profile of pontine proliferation. A: Raw data for proliferating cell density (#Ki671 cells/area of interest [mm2]) for each pons specimen, expressed as the total and region-specific (basis, tegmentum) densities. Age Groups I–V refer to pooled data used for time-dependent analyses of proliferation density detailed in B–E. B: Pons proliferation as a function of postnatal age, indicating a mono- phasic pattern of proliferation, with an 10-fold reduction between the first month of life (I) and 2–7 months of age (II). The proliferating cell density decreased significantly with increasing age (one-way ANOVA; F4,5 5 49.77, P < 0.001), and a significantly higher density of Ki671 cells was observed in the 0–1 months group (I, 28.1 cells/mm2) compared to all other groups (Tukey post-hoc comparison; P < 0.001). C: Proliferation decreased to very low levels by 1.5–2 years and remained very low throughout childhood (note second graph on right with reduced density scale on y-axis for time categories III–V). Note that a few Ki671 cells are found in the pons in late childhood and adolescence. D,E: Compartmental analysis of proliferation as a function of time and location revealed decreased proliferation with increasing age and in the tegmentum relative to basis (two-way ANOVA; time: F4,10 5 49.01, P < 0.001; location: F1,10 5 7.52, P < 0.05; time*location: F4,10 5 8.32, P 5 0.003). Proliferation was higher at 0–1 months (I) compared to all other timepoints (II–V) for both tegmentum (P < 0.02) and basis (P < 0.001) (Tukey post-hoc comparison). In addition, during the 0–1 months period (I) proliferation was higher in the basis relative to tegmentum (P < 0.001). At later timepoints examined in the subregion analysis (II–V), proliferating cell density within and between the two pons regions was not statistically different. immunohistochemistry suggested that increased myeli- ples ranging from birth to 15 years of age (Table 1). nation may partially account for this growth, particularly The density of proliferating cells in the entire pons, during the first 6 months of life. In order to determine defined as the number of Ki671 cells per mm2, was the contribution of cell proliferation to pontine growth, determined in 20 lm transverse sections (Fig. 4A). The we used 10 postmortem fixed human brainstem sam- proliferating cell density decreased significantly with

456 The Journal of Comparative Neurology | Research in Systems Neuroscience Human postnatal pons development

(P < 0.02) and basis (P < 0.001). In addition, at 0–1 months, proliferation was higher (P < 0.001) in the basis (36.1 cells/mm2) compared to tegmentum (15.5 cells/mm2). At the later four timepoints examined in the subregion analysis (Groups II–V), proliferating cell density within and between the two pons regions was not statistically different. In order to detect possible foci of proliferation that may be overlooked by counting Ki671 cells throughout the pons, we constructed spatial maps to assess the distribution of proliferation within the pons as a function of postnatal age (Fig. 5). In samples from 1-day brains, a higher density of proliferating cells was seen throughout the pons as compared to later timepoints. In addition, there was an increased density of Ki671 cells within the basis compared to the tegmentum, particularly in the ventral and lateral basis. Interestingly, at this very early postnatal age, Ki671 cells were observed in clusters within white matter tracts (cerebellar peduncle, corticospinal tracts, and transverse pontine fibers). Higher levels of proliferation were also noted throughout the pons at 1 month, with corresponding increased Ki671 cell density within the basis relative to tegmentum. However, the proliferating cells were distributed in a more homogeneous manner. At 7 months, Figure 5. Regional profile of pontine cell proliferation. Mapping of the number of Ki671 cells was markedly lower, and the 1 Ki67 cells within pons hemisections at different ages shows relative density of proliferating cells was similar in the decreased proliferation levels over time and a transition from a basis and tegmentum. In all brainstem samples ana- ventrally skewed distribution at 1 day to more homogeneous dis- 1 tributions at later timepoints. Note areas of increased density at lyzed from ages 1.5–13 years, Ki67 cells were rare 1 day corresponding to maturing white matter regions: middle and distributed throughout the basis and the tegmen- cerebellar peduncle (open arrowhead), transverse pontocerebellar tum. The above results suggest abundant cell prolifera- fibers (open arrow), and descending longitudinal corticospinal tion during the first 7 months after birth, with fiber tracts (solid arrow). D 5 dorsal, V 5 ventral, M 5 medial, L particularly high numbers during the first month. In 5 lateral. order to address the possibility that the observed proliferative cells might be reactive astrocytes, we performed increasing age (one-way ANOVA; F4,5 5 49.77, P < co-immunofluorescence for Ki67 and the astrocyte 0.001), and a significantly higher density of Ki671 cells marker GFAP (Fig. 6F). Confocal microscopy demon- was observed in the 0–1 months group (I, 28.1 cells/ strated that GFAP1 processes did not surround Ki671 mm2) compared to all other groups (Tukey post-hoc nuclei. In fact, we observed no reactive astrocytes in comparison; P < 0.001). In the 2–7 months group (II), our autopsy samples, but rather a network of normal, the density of Ki671 was reduced 10-fold (2.6 cells/ thin GFAP1 astrocyte processes throughout the tissue. mm2) compared to the 0–1 month group. From 2–13 years of age (Groups III–V), very low levels of proliferation were observed (0.09–0.17 cells/mm2) (Fig. 4B,C). Majority of postnatal proliferating cells are A similar analysis of proliferation was performed sep- Olig21 arately for the tegmentum and basis (Fig. 4D,E). A two- The MRI-based morphometric data showed robust way ANOVA revealed that proliferation density was a growth and increased myelination in the early postnatal function of time (F4,10 5 49.01, P < 0.001) and loca- pons. The histological analysis of pontine postmortem tion (F1,10 5 7.52, P < 0.05), and there was a signifi- tissue also showed cellular proliferation in the white cant interaction between time and location (F4,10 5 matter regions. These observations led us to analyze 8.32, P < 0.003). Tukey post-hoc analysis indicated the proportion of proliferating cells expressing Olig2, a that proliferation was higher at 0–1 months (Group I) progenitor marker that is frequently associated with the compared to all other timepoints for both tegmentum generation of oligodendrocytes. Human pons sections

The Journal of Comparative Neurology | Research in Systems Neuroscience 457 M.C. Tate et al.

Figure 6. Olig2/Ki67 immunohistochemistry. Confocal microscopy of sections from 1-day-old human mid-pons basis labeled with DAPI (A), Olig2 (B), Ki67 (C), and merged image (D) showing Ki671/Olig2– (solid arrow), Ki67–/Olig21 (open arrow), and Ki671/Olig21 (solid arrowhead) populations. E: Comparison of the percentage of Ki671 cells that were also Olig21 revealed that more than half of proliferative cells are double-positive and these cells were distributed throughout the pons; no significant effect of age or location was observed (two-way

ANOVA; time: F1,4 5 0.67, P 5 0.46; location F1,4 5 1.61, P 5 0.273). F: Confocal microscopy of section from pontine basis in 1-month sample labeled for Ki67 (red), GFAP (green), and DAPI (blue) illustrating that proliferative cells are not reactive astrocytes and do not colocalize with GFAP. Scale bars 5 20 lm. ranging from 1 day to 7 months were double-labeled The majority of Ki671 cells in the basis were Olig21 for Ki67 and Olig2, and the tegmentum and basis were (57.0 6 3.5%), but single-labeled cells for either marker analyzed (Fig. 6). In the 0–1 month group, double- were also observed (Fig. 6). By 2–7 months of age, the labeled Olig21/Ki671 cells were seen in both the basis number of Ki671 and Ki671/Olig21 cells declined in and the tegmentum. Increased density of both single- both pons locations relative to the 0–1 month group, labeled (Ki671) and double-labeled (Olig21/Ki671) consistent with the proliferation analysis described cells were observed in the basis relative to tegmentum. earlier. However, despite the smaller number of

458 The Journal of Comparative Neurology | Research in Systems Neuroscience Human postnatal pons development

Figure 7. Nestin/vimentin immunohistochemistry. Confocal images of three regions of the human pons labeled for nestin (green), vimentin (red), and DAPI (blue). A–D: At 1 postnatal day a dense population of vimentin1, nestin1, and vimentin1/nestin1 cells is observed in the ventricular zone of the dorsal pons. Within the tegmentum and basis, the few vimentin1/nestin1 processes were generally oriented parallel to white matter tracts. E–H: A markedly decreased VZ population of vimentin1/nestin1 cells can be identified in 7-month samples. The vimentin1/nestin1 processes seen in the tegmentum and basis within the first month of life were no longer seen by 7 months of age. I–L: By 8 years of age the nestin1 cells in the ventricular epithelium were no longer present, but a small subpopulation still expressed vimentin. Neither nestin1 nor vimentin1 cells were observed in 8-year-old basis or tegmentum. Scale bars 5 50 lm. proliferating cells in the 2–7 months age group, the pro- examined the spatial and temporal profile of nestin- portion of proliferating cells that were Olig21 remained and vimentin-expressing cells in the pons ventricular stable (61.8 6 2.57%) and was not statistically different zone, tegmentum, and basis. At postnatal day 1, a between pons subregions or when compared to 0–1 dense population of nestin1, vimentin1, and nestin1/ month data (Fig. 6E). vimentin1 cells was seen lining the ventricle (Fig. 7). Interestingly, the majority of these cells had radial processes into the tegmentum, suggesting the postnatal Nestin and vimentin expression in the persistence of a population of radial glia. At this early postnatal human pons timepoint, we also observed nestin1/vimentin1 proc- The above results indicate that more than half of the esses in both the tegmentum and basis. Interestingly, proliferating cells express Olig2. However, there was a in the basis these double-labeled processes were ori- significant fraction of Ki671 cells that were Olig2–.A ented parallel to white matter tracts. A similar pattern recent study describing nestin- and vimentin-positive of vimentin and nestin expression was observed at 1 cell populations in the pons throughout childhood and 2 months of age, although the total number of pos- (Monje et al., 2011) suggested that some such cells itive cells decreased. By 7 months, dorsal vimentin1/ may correspond to neural stem cells. We therefore nestin1 cells remained present in the ventricular region,

The Journal of Comparative Neurology | Research in Systems Neuroscience 459 M.C. Tate et al.

but only very rare single- or double-labeled cells were cal for the robust expansion of white matter tracts in observed in the parenchyma of the tegmentum or basis. this region during early postnatal life. While the source From 7 months through the later childhood years, this of this postnatal proliferative population in the pons is spatial restriction of nestin and vimentin expression to unclear, it raises the possibility that persistent primary the ventricular lining was maintained, although the num- progenitors in germinal niches of the postnatal brain- ber of vimentin1 radial fibers, and to a greater degree stem, analogous to the rhombic lip present during fetal the nestin1 processes, decreased over time. No spe- development, may continue to be a source of Olig21 cific labeling of nestin1 or vimentin1 processes was precursors. Alternatively, these precursors may be pro- seen in the basis or tegmentum at any ages over 7 duced before birth, and migrate widely to proliferate months. and differentiate locally. The present results are also relevant to better under- stand the origins of one of the most lethal types of DISCUSSION pediatric brain tumors. Diffuse intrinsic pontine glioma The present morphometric analysis of the human (DIPG) remains an important clinical problem, compris- postnatal pons demonstrates substantial growth of the ing 10–15% of childhood brain tumors (Fangusaro, pons throughout childhood, particularly in the basis 2009). Patients typically present at 5–9 years of age pontis. The MRI and histological studies suggest that and have a dismal prognosis, with a median survival of this expansion of the ventral pons is at least in part less than 1 year (Wagner et al., 2006). Genomic studies due to addition of oligodendrocytes and increased mye- have identified several mutations found in DIPG, includ- lination. This finding is consistent with previous immu- ing H3F3A/HIST1H3B (Wu et al., 2012), PDGFRA (Zar- nohistochemical and radiographic data showing that ghooni et al., 2010; Paugh et al., 2011; Puget et al., while myelination of the tegmentum is largely complete 2012), TP53 (Grill et al., 2012), and ADAM3A (Barrow by birth, an increase in myelination of the basis pontis et al., 2011). Recent work suggests that progenitor is observed in the first 3–4 months of life (Brody et al., cells give rise to other pediatric brain tumors, such as 1987; Kinney et al., 1988; Nozaki et al., 1992). At the cerebellar granule cell precursor-derived medulloblasto- cellular level, the wave of Olig21 cell proliferation mas (Schuller et al., 2008), precerebellar neuronal observed not only in the basis, but also to a lesser precursor-derived medulloblastomas (Gibson et al., degree in the tegmentum, appears to peak early in 2010), and radial glia-derived ependymomas (Taylor postnatal life (0–1 month). Yet the pons continues to et al., 2005). Given the predominant ventral growth of grow at high rates up to 1 year and at slower rates up DIPG (Fischbein et al., 1996) and a recent report of to 5 years of age. This suggests that only some of the expression of progenitor markers in the postnatal observed growth can be attributed to cell addition, and human pons (Monje et al., 2011), it has been suggested that the slow, steady growth observed from 1 to 5 that the ventral pons may contain the cell of origin for years is likely due to increase in the neuropil, including DIPG. In addition, a recent report demonstrated that expansion of the corticospinal tract. Consistent with >90% of DIPGs express Olig2 (Ballester et al., 2013). this finding, a previous study demonstrated a decrease These data, in combination with the findings in the in neuronal number and volume in the pontine nuclei of present study, suggest some interesting hypotheses. the basis between birth and 2 months (Nozaki et al., First, the presence of a dorsal vimentin1/nestin1 cell 1992). Our data suggest that most of the growth after population in the human pons throughout childhood 1 year is related to an increase in cellular processes suggests that this cell population may be a potential and cellular size rather than cell number. cell of origin for focal human brainstem tumors, which The tremendous growth of the human pons during tend to occur dorsally in the region of the aqueduct of infancy and childhood raises fundamental questions, Sylvius/4th ventricle in the midbrain, pons, and medulla not only of the basic cellular mechanisms that explain (Fischbein et al., 1996). Second, the scarcity of prolifer- this expansion, but also about its functional correlates. ative progenitors past early postnatal stages suggests Pontine growth is likely associated with consolidation of that if DIPG originates from a proliferating cell in the neural circuits for motor coordination and learned ventral pons, oncogenic events would likely occur early behaviors during this critical stage of human develop- in childhood, and it may take years for these tumors to ment, consistent with experimental data showing robust become fully malignant. By contrast, if DIPG originates synaptogenesis and consolidation of cortico-pontine shortly before diagnosis (ages 5–9), a time period tracts in the early postnatal time period (Adams et al., where we observed that pons growth is rapidly deceler- 1980; Mihailoff et al., 1984). The ventral pool of prolif- ating, then DIPG may derive from transformation of a erating Olig21 cells within the basis pontis may be criti- nonproliferative cell, for example, a Olig21/Ki67– cell.

460 The Journal of Comparative Neurology | Research in Systems Neuroscience Human postnatal pons development

We observed very low numbers of Ki671 cells in the technical, and material support: MT, RL, TN, EH. pons during the period when DIPG is most frequently Study supervision: AA-B, DR. diagnosed, although we cannot exclude that those rare dividing cells could be the origin of these tumors. LITERATURE CITED In summary, the present study provides a quantita- Abe S, Takagi K, Yamamoto T, Okuhata Y, Kato T. 2004. Semi- tive description of the extraordinary growth of the quantitative assessment of myelination using magnetic resonance imaging in normal fetal brains. Prenat Diagn human pons during infancy and childhood. Our data 24:352–357. indicate that both the tegmentum and the basis grow Adams CE, Parnavelas JG, Mihailoff GA, Woodward DJ. 1980. significantly during this period, but the basis pontis The neurons and their postnatal development in the basi- expands at rates higher than the tegmentum or the lar pontine nuclei of the rat. Brain Res Bull 5:277–283. Ballester LY, Wang Z, Shandilya S, Miettinen M, Burger PC, medulla. This preferential growth of the basis is at Eberhart CG, Rodriguez FJ, Raabe E, Nazarian J, Warren least in part mediated by increased myelination, con- K, Quezado MM. 2013. Morphologic characteristics and sistent with the presence of a large number of prolifer- immunohistochemical profile of diffuse intrinsic pontine 1 gliomas. Am J Surg Pathol 37:1357–1364. ating Olig2 cells during early postnatal ages. These Barkovich AJ, Millen KJ, Dobyns WB. 2009. A developmental data, in conjunction with previous studies of brainstem and genetic classification for midbrain-hindbrain malfor- development, suggest distinct cell populations as mations. Brain 132(Pt 12):3199–3230. potential cells of origin for different brainstem tumors. Barrow J, Adamowicz-Brice M, Cartmill M, MacArthur D, Lowe J, Robson K, Brundler MA, Walker DA, Coyle B, Grundy Future studies evaluating the origin and fate of post- R. 2011. Homozygous loss of ADAM3A revealed by natal progenitor populations in model systems may genome-wide analysis of pediatric high-grade glioma and give insight into the mechanisms of the postnatal diffuse intrinsic pontine gliomas. Neuro Oncol 13:212– 222. growth of the pons and a possible cell of origin of Bohn W, Wiegers W, Beuttenmuller M, Traub P. 1992. Spe- pediatric pontine gliomas. cies-specific recognition patterns of monoclonal antibodies directed against vimentin. Exp Cell Res 201:1–7. Brody BA, Kinney HC, Kloman AS, Gilles FH. 1987. Sequence ACKNOWLEDGMENTS of central nervous system myelination in human infancy. M.T. was supported by the UCSF Neurological Surgery I. An autopsy study of myelination. J Neuropathol Exp Residency Program. R.L. is supported by the UCSF Medi- Neurol 46:283–301. cal Scientist Training Program, Neuroscience Graduate Debus E, Weber K, Osborn M. 1983. Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for Program, and Discovery Fellows Program. D.H.R. is an each of the neurofilament triplet polypeptides. Differen- HHMI Investigator. A.A.-B. is the Heather and Melanie tiation 25:193–203. Muss Endowed Chair and Professor of Neurological Essick CR. 1912. The development of the nuclei pontis and the nucleus arcuatus in man. Am J Anat 13:25–54. Surgery. Procurement of pediatric brain tissue at UCSF Fangusaro J. 2009. Pediatric high-grade gliomas and diffuse was supported by University of California MRPI grant intrinsic pontine gliomas. J Child Neurol 24:1409–1417. #142675 to E.J.H. The authors thank Jeanelle Ariza of the Fischbein NJ, Prados MD, Wara W, Russo C, Edwards MS, Pediatric Neuropathology Research Laboratory (UCSF, Barkovich AJ. 1996. Radiologic classification of brain stem tumors: correlation of magnetic resonance imaging HHMI) for assistance with myelin staining and the labora- appearance with clinical outcome. Pediatr Neurosurg 24: tories of Drs. Arnold Kriegstein and Patrick McQuillen for 9–23. assistance with automated microscopy techniques. Gibson P, Tong Y, Robinson G, Thompson MC, Currle DS, Eden C, Kranenburg TA, Hogg T, Poppleton H, Martin J, Finkelstein D, Pounds S, Weiss A, Patay Z, Scoggins M, CONFLICT OF INTEREST Ogg R, Pei Y, Yang ZJ, Brun S, Lee Y, Zindy F, Lindsey JC, Taketo MM, Boop FA, Sanford RA, Gajjar A, Clifford The authors report no conflicts of interest. SC, Roussel MF, McKinnon PJ, Gutmann DH, Ellison DW, Wechsler-Reya R, Gilbertson RJ. 2010. Subtypes of medulloblastoma have distinct developmental origins. ROLE OF AUTHORS Nature 468:1095–1099. Allauthorshadfullaccesstoallthedatainthe Grill J, Puget S, Andreiuolo F, Philippe C, MacConaill L, Kieran MW. 2012. Critical oncogenic mutations in newly diag- study and take responsibility for the integrity of the nosed pediatric diffuse intrinsic pontine glioma. Pediatr data and the accuracy of the data analysis. Study Blood Cancer 58:489–491. concept and design: MT, RL, NS, DR, AA-B. Acquisi- Hatta T, Satow F, Hatta J, Hashimoto R, Udagawa J, Matsumoto A, Otani H. 2007. Development of the pons tion of data: MT, RL, TN, AB. Analysis and interpreta- in human fetuses. Congenit Anom (Kyoto) 47:63–67. tion of data: MT, RL, TN, AB, EH, DR, AA-B. Drafting Kinney HC, Brody BA, Kloman AS, Gilles FH. 1988. Sequence of the article: MT, RL, TN. Critical revision of the arti- of central nervous system myelination in human infancy. cle for important intellectual content: MT, RL, TN, NS, II. Patterns of myelination in autopsied infants. J Neuropathol Exp Neurol 47:217–234. AB,EH,DR,AA-B.Statisticalanalysis:MT,RL. Ligon KL, Alberta JA, Kho AT, Weiss J, Kwaan MR, Nutt CL, Obtained funding: MT, EH, DR,AA-B.Administrative, Louis DN, Stiles CD, Rowitch DH. 2004. The

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